The paracellular route is the dominant pathway for passive solute flow across the intestinal epithelial barrier, and its permeability depends on the regulation of intercellular tight junctions (tj), also known as the zonula occludens (ZO). A century ago, these structures were thought to be a secreted extracellular cement forming an absolute and unregulated barrier within the paracellular space. Biological studies of the past several decades have shown that tj are dynamic structures whose physiological regulation remains largely undefined. In our original grant, we used zonula occludens toxin (Zot), a protein elaborated by Vibrio cholerae, as a tool to study the regulation of tj function. The results obtained during the 3 years of funding demonstrated that Zot reversibly opens tj by interacting with a specific surface receptor(s) coupled to a complex intracellular signaling pathway involving PKCalpha-dependent polymerization of actin monomers into actin microfilaments strategically localized to regulate the paracellular pathway. Furthermore, we were able to purify and partially characterize a human eukaryotic Zot analogue, that we named zonulin, that represents a novel, endogenous modulator of tj function. The long term objectives of the proposed application are to further define the mechanism(s) of action of both Zot and zonulin and to purify and clone the Zot surface receptor to gain insights into tj regulation, particularly at the cellular and molecular levels.